Genetic population dynamics of the critically endangered scalloped hammerhead shark (Sphyrna lewini) in the Eastern Tropical Pacific

Abstract The scalloped hammerhead shark, Sphyrna lewini, is a Critically Endangered, migratory species known for its tendency to form iconic and visually spectacular large aggregations. Herein, we investigated the population genetic dynamics of the scalloped hammerhead across much of its distribution in the Eastern Tropical Pacific (ETP), ranging from Costa Rica to Ecuador, focusing on young‐of‐year animals from putative coastal nursery areas and adult females from seasonal aggregations that form in the northern Galápagos Islands. Nuclear microsatellites and partial mitochondrial control region sequences showed little evidence of population structure suggesting that scalloped hammerheads in this ETP region comprise a single genetic stock. Galápagos aggregations of adults were not comprised of related individuals, suggesting that kinship does not play a role in the formation of the repeated, annual gatherings at these remote offshore locations. Despite high levels of fisheries exploitation of this species in the ETP, the adult scalloped hammerheads here showed greater genetic diversity compared with adult conspecifics from other parts of the species' global distribution. A phylogeographic analysis of available, globally sourced, mitochondrial control region sequence data (n = 1818 sequences) revealed that scalloped hammerheads comprise three distinct matrilines corresponding to the three major world ocean basins, highlighting the need for conservation of these evolutionarily unique lineages. This study provides the first view of the genetic properties of a scalloped hammerhead aggregation, and the largest sample size‐based investigation of population structure and phylogeography of this species in the ETP to date.


| INTRODUC TI ON
Populations of many oceanic shark and ray species have declined dramatically since the onset of industrial fishing, with three-quarters of these large-bodied species facing increased risk of extinction, mainly from over-exploitation (Pacoureau et al., 2021). Knowledge of the population dynamics of these oceanic sharks is required for guiding urgently needed, science-based conservation management efforts, and understanding the ecology and evolutionary biology of these high trophic-level marine predators.
Delineating management units (sensu Moritz, 1994) of oceanic shark species is made complex by their high vagility (Musick et al., 2004) and can be informed by an understanding of population genetic connectivity, including the influencing roles of dispersal and philopatry. Regional philopatry, as defined by Chapman et al. (2015), describes a highly mobile, roaming individual that typically returns to the region of its birth to reproduce, thereby limiting and/or restricting gene flow to within a much smaller geographic area than would otherwise be expected based on the vagility of the species alone.
Nevertheless, given the migratory propensity of oceanic sharks, genetic differences among discrete reproductive units may be obscured by sampling and testing for differentiation among adults during non-reproductive periods or by pooling samples across age classes, as varying life-stages possess varying dispersal tendencies (Klein et al., 2019;McClain et al., 2022;Phillips et al., 2021). For instance, following parturition, many young-of-year (YOY) or juvenile sharks remain within coastal nursery habitats for many months, as these habitats may serve as protection from predators or may be a location where there is an abundance of prey, whereas, in contrast, older life stages may disperse for feeding, reproduction, and other social behaviors. Thus, sampling highly vagile species of elasmobranchs at YOY stages or females undergoing parturition will likely provide the most useful information concerning genetic connectivity and how best to identify genetic management units.
Scalloped hammerheads are also noted for forming seasonal aggregations at offshore oceanic islands and seamounts in some parts of its distribution, possibly to facilitate social interactions, utilize cleaning stations, mate, and/or use as a staging location from which to conduct nocturnal foraging excursions into the surrounding pelagic environment (Bessudo et al., 2011;Brown et al., 2016;Hearn et al., 2010;Klimley & Nelson, 1984;Salinas-de-León et al., 2017).
The Critically Endangered status (IUCN Red List; Rigby et al., 2019) of the scalloped hammerhead globally has resulted in several broadscale conservation policy measures (e.g., Appendix II listings on the Convention on International Trade in Endangered Species (CITES) and Convention on the Conservation of Migratory Species of Wild Animals), but this species is still harvested and traded for its meat and fins legally and illegally worldwide (Abercrombie et al., 2005;Rigby et al., 2019). The exploitation of scalloped hammerheads is particularly problematic in the Eastern Tropical Pacific (ETP), a highly biodiverse biogeographic region ranging from southern Mexico to northern Peru, including the Galápagos archipelago, where illegal, unreported and unregulated (IUU) fishing is widespread Enright et al., 2021;Espinoza et al., 2018).
Within the ETP, scalloped hammerheads form aggregations at offshore islands, including the northern Galápagos Islands of Darwin and Wolf, Malpelo Island (Colombia) and Cocos Island (Costa Rica) (Bessudo et al., 2011;Hearn et al., 2010;Nalesso et al., 2019), all designated as World Heritage Sites (UNESCO, 2021). The seasonal aggregations that form in the Galápagos Islands number in the few thousands and are composed mainly of adult females, many of whom are thought to be pregnant based on their expanded girth (Hearn et al., 2010;Ketchum et al., 2014;Salinas-de-León et al., 2016. To preserve this aggregation and other species in the waters of the Galápagos, the Ecuadorian government established the Galápagos Marine Reserve (GMR) and prohibited all shark fishing and landing F I G U R E 1 Scalloped hammerhead shark, Sphyrna lewini, in the Galápagos. Copyright: Pelayo Salinas-de-León.
within the GMR (Carr et al., 2013). However, scalloped hammerheads in the Galápagos are migratory, moving outside the bounds of the GMR into international waters where they face intense pressure from IUU fisheries Carr et al., 2013;Dulvy et al., 2008). Notably, a temporal decline in abundance of females at the Galápagos aggregation site coincides with the appearance of YOY sharks in ETP mainland coastal nursery habitats (Nalesso et al., 2019) and recent telemetry work has documented adult female dispersal linking these two regions (P. Salinas de León and M. Shivji, unpublished). Furthermore, a direct parent-offspring genetic connection between female scalloped hammerheads at Malpelo Island and YOY sharks in coastal nursery sites in Colombia was found (Quintanilla et al., 2015). These observations support the hypothesis that females in the Galápagos aggregations are using mainland coastal sites for parturition.
Understanding the population genetic dynamics and genetic diversity of scalloped hammerhead aggregations, and of this critically endangered shark across its ETP distribution, can provide insight into its biology, genetic health and resilience, and is of conservation management relevance Thomson et al., 2021).
Eight previous studies examining the population genetic structure and phylogeography of scalloped hammerheads, from local to global scales, have included samples from at least one location in the broader eastern Pacific (Castillo-Olguín et al., 2012;Daly-Engel et al., 2012;Green et al., 2022;Nance et al., 2011;Quintanilla et al., 2015;Rangel-Morales et al., 2022;Villate-Moreno et al., 2022). These studies have added to the body of knowledge about this species in this region, but their inferences have been constrained by either samples obtained from only one or a few sites, small samples sizes collected opportunistically from fisheries landings (given difficulties of sampling threatened megafauna), and in some cases sample sets pooled across variable demographic groups (YOY, juveniles, adults), factors which can lead to erroneous conclusions about population structure and dispersal patterns (McClain et al., 2022;Phillips et al., 2021). This previous work has offered inconsistent evidence of female philopatry in scalloped hammerheads within the ETP (e.g., Castillo-Olguín et al., 2012;Daly-Engel et al., 2012;Nance et al., 2011;Rangel-Morales et al., 2022), despite this behavior having been suggested elsewhere globally (Chapman et al., 2009;Daly-Engel et al., 2012;Pinhal et al., 2020). Nevertheless, most of these previous studies indicate that gene flow throughout the ETP is at least somewhat restricted; however, at what spatial scale and whether this genetic differentiation is driven by female philopatry across the region remains unclear, thus warranting further investigation.
To add to existing information on scalloped hammerheads in the ETP, here we address four objectives. We: (1) assess the genetic diversity specifically of adult scalloped hammerheads from the Galápagos aggregations and adult sharks sampled from two other global regions to compare their genetic health and potential resiliency; (2) test for the presence of regional philopatry by ETP scalloped hammerheads by analyzing for population structure in YOY sharks collected from nursery sites and for relatedness in YOY within and among nursery sites; (3) analyze genetic kinship to determine whether (a) relatedness may be driving aggregation behavior in Galápagos adults and (b) the Galápagos aggregation adult females are using mainland coastal nursery sites for parturition; and (4) (Table 1). Samples from only adult sharks (GAL, SEY and FLA locations) were used in some of the subsequently described analyses to allow genetic comparison across equivalent age demographic groups.
All tissue samples were stored in 95% undenatured ethanol.
Genomic DNA was extracted using Qiagen DNeasy Blood and Tissue Kits according to the manufacturer's instructions (Qiagen Inc.).

PCR reactions were carried out on an Applied Biosystems BioRad™
Thermal Cycler with the following thermal profile: 95°C for 15 min, 35 cycles of 94°C for 1 min, 1 min at the primer-specific annealing temperature (i.e., TA = 50°C for Cli-12, TA = 56°C for SMO7, TA = 60°C for SLE045, SLE089, SMO3, SMO8, and TA = 65°C for SLE018, SLE027, SLE033, SLE038), and 72°C for 2 min, followed by a final extension of 72°C for 20 min. Electrophoresis of amplified microsatellite loci was performed on an Applied Biosystems 3130 Genetic Analyzer. Alleles were sized using GeneScan LIZ 600 size standard and scored using the software GeneMapper v.3.7 (Applied Biosystems Inc.). Electropherograms were visually inspected by two researchers, and samples genotyped at fewer than seven microsatellite loci were discarded.
To ensure that no sample duplicates (e.g., repeated sampling of the same adults or YOYs) were included in downstream analyses, match analysis was performed as implemented in the Excel Microsatellite Toolkit (Park, 2001). Pairs of individuals possessing two or less mismatched alleles were considered likely duplicates (two mismatched alleles were allowed to account for possible genotyping error), and where matches were found, one multi-locus genotype (along with its corresponding haplotype if present within the mitochondrial control region dataset) per putative duplicate pair was discarded. We tested for genotyping errors, null alleles, large-allele dropout and stutter using Microchecker 2.2.3 (Van Oosterhout et al., 2004), and used FreeNa (Chapuis & Estoup, 2007) to directly estimate the frequency of null alleles with 1000 iterations. All loci were checked for subpopulation-level deviations from Hardy-Weinberg Equilibrium (HWE) and linkage disequilibrium (LD) using GENEPOP on the Web F I G U R E 2 Sampling sites (13 red circles) and sample sizes (in brackets next to circles) of Sphyrna lewini in the Eastern Tropical Pacific. Black numbers in brackets represent the number of shark samples microsatellite genotyped at each location. Blue numbers in brackets represent the number of mitochondrial control region sequences from each location obtained in this study. Red numbers in brackets represent the number of mitochondrial control region sequences obtained from Quintanilla et al. (2015) and pooled with sequences from this study for analyses. Asterisks indicate locations of adult female samples. All other location samples are YOY (young-of-year) animals. Subpopulation abbreviations: BPA, Bahía Parita; BVA, Buenaventura; DAR, Darwin Arch; COJ, Cojimies; GNI, Golfo Nicoya; GDU, Golfo Dulce; GMO, Golfo de Montijo; TRI, Tribugá; MAL, Malpelo Island; SAN, Sanquianga; SEC, South Ecuador; SGA, South Galápagos; WOL, Wolf Island. (Rousset, 2008), with significance of deviations estimated using the Markov chain method and estimated probabilities corrected for multiple comparisons in R (p.adjust; R Core Team, 2020) with the false discovery rate (FDR) (Benjamini & Hochberg, 1995). Problematic loci, i.e., those with high levels of null alleles and/or deviations from HWE within more than three subpopulations, were excluded from downstream analysis. The statistical power of the suite of microsatellites given different levels of F ST was estimated using POWSIM 4.1 (Ryman & Palm, 2006), assuming an effective population size (N e ) of 500. Final POWSIM estimates were derived from 100 simulations per run, and Fisher's exact test analyses implemented 1000 dememorizations, 100 batches, and 1000 iterations per batch.

| Microsatellite DNA: Analysis of population genetic structure within the ETP and among global adults
Microsatellite summary statistics [number of alleles (A), allelic richness (A R ), inbreeding coefficient (F IS )] were determined for each locus and subpopulation using the program FSTAT 2.9.4 (1000 iterations, Goudet, 2001), while expected and observed heterozygosities (H E and H O ; Nei, 1978) were estimated with GenAlEx 6.5 (Peakall & Smouse, 2012).
To test for nuclear genetic population structure among scalloped hammerhead subpopulations in the ETP and among the globally sampled adults, we adopted both pairwise and cluster-based analyses.
First, pairwise nuclear genetic differentiation was assessed among: (1) adult shark subpopulations (SEY, FLA, and GAL) and (2) all ETP subpopulations (DAR, WOL and all YOY subpopulations). Pairwise metrics F ST (Weir & Cockerham, 1984), standardized G ′′ ST (Meirmans & Hedrick, 2011), and Jost's D EST (Jost, 2008) were estimated for all comparisons using GenAlEx; significance of values was determined using 999 permutations and estimated probabilities were adjusted with the FDR. Second, to test for isolation-by-distance (IBD; Bohonak, 2002) among ETP mainland putative nursery YOY subpopulations, a Mantel Test was performed in GenAlEx. Geographic distances were estimated between geographic coordinates using GenAlEx and significance of the correlation determined with 999 permutations. And third, nuclear microsatellite differentiation was further investigated using adegenet 2.1.5 (Jombart, 2008)   . Clusters were pre-assigned based on a priori subpopulations and all discriminant functions (DAs) were retained. The optimal number of principal components to include for each DAPC was determined using α-score validation. DAPC outcomes were visualized as a scatterplot of genetic distance between groups.

| Microsatellite DNA: Assessment of relatedness-Galápagos aggregation adults and ETP Young of Year sharks
We tested for relatedness and familial relationships in ETP scalloped hammerheads using a multi-tiered approach. Potential parent-offspring relationships among adult female sharks from the Galápagos aggregations and YOY sharks sampled from ETP nurseries were assessed using the programs Cervus 3.0.7 (Marshall et al., 1998) and Colony 2.0 (Jones & Wang, 2010). Cervus implements a pairwise likelihood-based approach to assign offspring to the most likely true parent from a pool of candidate parents. Cervus was run three times with the parameter "proportion of candidate parents sampled" set to 0.1, 0.01, and 0.001.
Parentage and sibling relationships (full-and half-siblings) among ETP sharks were assessed using Colony 2.0, which uses pedigree reconstruction to infer likelihood of genetic relationships, rather than a pairwise approach. Analyses were performed assuming female and male polygamy (per Marie et al., 2019), dioecious and diploid samples, a genotyping error rate of 0.01, and an absence of inbreeding or clones. All analyses were performed assuming the Full-Likelihood (FL) method with high precision, sibship scaling, no updating of allele frequencies, and a weak sibship prior. Three runs were performed using the "very long" option in the program (with three different random number seeds) to ensure consistency of familial assignments; and for comparison purposes, three additional Colony runs were performed assuming duplicate parameters as outlined above, however, these runs assumed the presence of inbreeding. Relationships were only deemed "true" if parent-offspring or sibling pairs were identified across all six Colony runs with >95% probability and were deemed "possible" if probabilities exceeded 90%. To support any inferred parent-offspring and/or sibling relationships: (1) parentage exclusion probabilities were estimated using the program COANCESTRY 1.0.1.10 (Wang, 2011) and all adult Galápagos genotypes, and by assuming a genotyping error rate of 0.01 and (2) by estimating pairwise relatedness among all ETP sharks using COANCESTRY. To determine the most appropriate relatedness estimator for our dataset, we used the R package related (Pew et al., 2015) to simulate 1000 pairs of individuals for each of four relatedness groups (parent-offspring, full-siblings, half-siblings, and unrelated) and four relatedness metrics (i.e., Li et al., 1993;Queller & Goodnight, 1989;Lynch & Ritland, 1999;Wang, 2002), and using ETP scalloped hammerhead microsatellite allele frequencies. Within the simulations, Wang (2002) possessed the highest correlation between the observed and expected relatedness values (r 2 = 0.82) and was therefore selected for use herein (data not shown). Overall mean relatedness (Wang, 2002) among individuals was calculated for combined ETP adults and YOYs. For each pair of colony-identified putative parent-offspring pairs, or full-or half-sibling pairs, pairwise relatedness was estimated for comparison to the overall ETP mean value (see "Section 3").

| Mitochondrial control region sequencing and published data mining
The complete mitochondrial DNA control region (~1200-bp) was am- control region studies (Chapman et al., 2009;Nance et al., 2011;Quintanilla et al., 2015). Prior to downstream population genetic analysis, species identity of all sequences was tested using the National Center for Biotechnology Information BLAST tool (Altschul et al., 1990), and any identified species misidentifications were discarded (along with its corresponding multilocus genotype if present within the microsatellite dataset).
To increase the mitochondrial sequence dataset for our ETP analyses, we added 63 published sequences (Quintanilla et al., 2015) of scalloped hammerheads sampled from the Colombian Pacific

| Microsatellite genotyping and mitochondrial control region sequencing-quality control and filtering
Across the entire genotyped dataset, six pairs of likely duplicate samples were found (i.e., individuals shared the same microsatellite profile save for two or less mismatched alleles); five of these six putative pairs were comprised of individuals sampled from the same geographic location and had the same mitochondrial control region haplotype, suggesting inadvertent duplicate sampling of YOY market-derived sharks (no duplicates were found among adult samples). A single individual from each of these five pairs (i.e., the individual with the highest rate of missing data) was discarded from both control region and microsatellite datasets. The individuals comprising the sixth pair, however, were collected from different locations and processed at separate times in the laboratory so both were re-  The final percentage of missing data for the nine-locus microsatellite dataset was 5.55%. Loci were polymorphic in all locations and the number of alleles per locus ranged from 6-52 (Table S1) Table 2), along with strong phylogeographic structure and no haplotype sharing among sampling sites ( Figure 4a). However, in contrast to the microsatellite patterns of geographic genetic differentiation (Figure 3a), the mitochondrial network indicated that recovered SEY and FLA haplotypes were more closely evolutionarily related than the SEY and GAL haplotypes (SEY and FLA clades were separated by only a single mutational step; the most closely related haplotypes between SEY and GAL were separated by 18 unsampled mutational steps).

| Microsatellites
The overall microsatellite diversity estimates from nine loci (av-  Table 2), and DAPC cluster analysis (34 PCs retained) also did not reveal any distinct separation and clustering of multi-locus genotypes (Figure 3b).
Microsatellite IBD analysis found no correlation between genetic and geographic distance among coastal mainland YOY nurseries (r = 0.272, p = .127).

| Mitochondrial DNA
Analysis of 478 control region sequences from 13 ETP locations (Adult and YOYs; Figure 2) yielded overall haplotype and nucleotide diversity estimates of 0.524 ± 0.016 and 0.001 ± 0.001, respectively.
In addition, mitochondrial diversity estimates were largely similar among the ETP adult and YOY sampling sites (   consistently identified across all six "very long" Colony runs with a probability exceeding 95%; however, one "possible" full-sibling pair was identified in five of six runs at 91% probability (Table 3).

| Relatedness of scalloped hammerheads within aggregations and nursery sites
Pairwise relatedness (Wang, 2002) of this "possible" adult pair was  Among YOY ETP samples, Colony identified four full-sibling pairs (comprising eight separate individuals) that were consistently detected across the six "very long" runs with probabilities greater than 95% (and two additional "possible" full-siblings with probabilities >90%). Each of these pairs possessed pairwise estimates of relatedness that were higher than the overall mean value (Table 3), and three of the four full-sibling pairs (>95%) were sampled from the same nursery sites within a single day of each other; all sibling pairs contained matching mitochondrial haplotypes. Notably, two of these pairs were sampled from the COJ nursery, one pair from the SEC nursery, and one pair contained individuals sampled from COJ and SEC. An additional two "possible" half-sibling pairs-each with 90-91.2% probability across all six runs and higher than average pairwise relatedness estimates (Table 3)  ; Northwest Indian (samples grouped from: Arabian Sea and Red Sea; Spaet et al., 2015); Southwest Indian (samples grouped from: Seychelles and South Africa;  this study); East Indian (samples grouped from: West Australia, Indonesia, and Thailand; Green et al., 2022); Western North Pacific (samples grouped from: Philippines and Taiwan; ; Northern Territory (N. Australia) (Green et al., 2022); Western South Pacific (samples grouped from: Papua New Guinea, East Australia, Princess Charlotte Bay, Townsville, New South Wales, and Fiji; Green et al., 2022); Central Pacific (Hawai'i; ; Mexican Pacific (samples grouped from: Baja California, La Paz, and Mazatlan; Nance et al., 2011); Eastern Tropical Pacific (samples grouped from: Costa Rica (Tarcoles, Golfo de Nicoya, Golfo Dulce), Panama (Pacific Panama, Golfo de Montijo, Chiriqui, Bahia de Parita), Colombia (Tribugá, Utria, Malpelo Island, Buenaventura, Sanquianga), Ecuador (Cojimies, South, Manta); Nance et al., 2011;Quintanilla et al., 2015; this study); Galápagos (Darwin Arch, Wolf Island, South; this study).

TA B L E 3
Sphyrna lewini Eastern Tropical Pacific full-and half-sibling pairs identified at probabilities greater than or equal to 90% across any of the six "very long" runs, along with the sibling pair's estimated pairwise relatedness per Wang (2002).

| Worldwide matrilineal phylogeography
A median-joining network of scalloped hammerhead control region haplotype sequences (515 bp) from all age groups across 12 worldwide locations (i.e., Worldwide-CR-Dataset), illustrated three primary phylogeographic lineages consisting of samples from the: (1) Atlantic, (2) western Indian, and (3) eastern Indian-Pacific regions with minor haplotype sharing between the latter two ( Figure 4b).
No phylogeographic partitioning was detected among haplotypes within the largely Pacific clade, with widespread sharing of the most common haplotypes occurring across the Pacific. Within the Atlantic, there was some separation between the western North and western South Atlantic, albeit with some haplotype sharing. The eastern Atlantic is nested within the greater Atlantic clade, however no haplotype sharing between the western and eastern Atlantic was observed.

| DISCUSS ION
This study complements and expands on previous studies of scal- In contrast to the mitochondrial DNA results, nuclear differentiation between the Seychelles and Galápagos adults was much smaller than between these two populations and Florida adults.
This contrast in differentiation between the two organelle markers may be attributed to their mutation rate and mode of inheritance.
Biparentally inherited microsatellites mutate faster than matrilineally inherited mtDNA, and can be used to detect population structure, or conversely male-mediated gene flow, on a relatively contemporary timescale (10-100 generations ago) (Selkoe & Toonen, 2006).  Ovenden et al., 2011;Daly-Engel et al., 2012;Spaet et al., 2015;Pinhal et al., 2020;Green et al., 2022), lending further support to the notion that genetic diversity in the ETP still remains comparatively high. Notably, the average H O values for ETP scalloped hammerheads in our study were also higher than average H O values of 20 of the 28 shark species reviewed by Domingues et al. (2018).

Microsatellite
Our findings of little genetic differentiation among the scalloped hammerhead adults and YOY in the ETP, coupled with geographically widespread mitochondrial control region haplotype sharing and no evidence of IBD among YOY sampled along the mainland putative nurseries, supports the presence of high genetic connectivity in this region, and is inconsistent with a hypothesis of philopatric behavior by females in this region. Rather, our data are consistent with studies suggesting females of this species stray between coastally connected nursery areas for parturition Quintanilla et al., 2015).  (Costello et al., 2017), whereas the ETP is considered a single biogeographic realm with few geographic barriers to gene flow (Costello et al., 2017;Floeter et al., 2008;Kulbicki et al., 2013).

| Relatedness of scalloped hammerheads within aggregations and nursery sites
The northern Galápagos Islands boast the highest shark biomass per area in the world, with more than half of this biomass compris- Atlantic demonstrated that within group relatedness was higher than expected by chance, especially among the females (Lieber et al., 2020). In contrast, Venables et al. (2021) found no evidence that reef manta rays (Mobula alfredi) aggregating at sites in the western Indian Ocean were more related than expected by chance and suggested that kinship did not play a role in visits to aggregation sites. Furthermore, no correlation between kinship and social networks (although not technically aggregations) was found in blacktip reef sharks (Carcharhinus melanopterus) in French Polynesia (Mourier & Planes, 2021). In our study of the aggregating hammerhead adults, only a single "possible" full-sibling and no half-sibling pairs were identified, suggesting that relatedness is not a driver of how individuals choose the Wolf and Darwin Islands aggregation sites.
Some shark species create cognitive maps using the earth's magnetic field to navigate back to locations with prey availability (Keller et al., 2021;Kimber et al., 2014;Meyer et al., 2010), and scalloped hammerheads are hypothesized to use geomagnetic topotaxis for navigation between seamounts (Klimley, 1993). It is possible that once a topographically and environmentally suitable insular aggregation site is discovered during adult migrations, the scalloped hammerheads remember and repeatedly return to the aggregation site for the social interaction and/or foraging benefits it provides.
Unlike Quintanilla et al. (2015)  Although sample sizes analyzed in this study were the largest from the ETP to date, the number of sharks present in the aggregations and nursery sites is not known but is likely to be large based on the high frequency of YOY sharks found in artisanal markets (Guzman et al., 2020;O'Bryhim et al., 2021). It is possible, therefore, that the failure to find parent-offspring pairs in our study is a result of insufficient sampling of YOY sharks. Likewise, we found only four high probability full-siblings and no-half sibling pairs across 289 genotyped YOYs. While kinship results in this study provide a preliminary view of the reproductive behavior of scalloped hammerheads in the ETP, further insight will require more exhaustive sampling of YOYs and the use of a larger marker set.

| Worldwide matrilineal phylogeography
To add to existing matrilineal phylogeographic hypotheses about scalloped hammerheads worldwide Fields et al., 2020;Green et al., 2022;Quintanilla et al., 2015;Spaet et al., 2015), we added our 515 mtCR sequences (from the ETP, Seychelles and Florida) to published data from other global collection sites increasing the global dataset by almost a third, thus providing a phylogeographic view based on the largest dataset to date (1818 individual sequences). Scalloped hammerheads clustered primarily into three phylogeographic lineages corresponding to the Atlantic, western Indian, and eastern Indian-Pacific regions with minor haplotype sharing between the latter two. The observed pattern of ocean basin lineage relationships derived from this much larger sequence dataset remain concordant with previous hypotheses of closer evolutionary relationships between scalloped hammerheads in the Atlantic and Indian Ocean, relative to the Pacific lineage. We note that the addition of 415 sequences from the ETP revealed haplotype sharing between scalloped hammerheads from the western and eastern Pacific, as one of the most common haplotypes is represented in all Pacific sampling locations. Although multiple low frequency haplotypes were found to be unique to the ETP, more exhaustive sampling of the western Pacific is needed to determine whether these haplotypes are present but unsampled in other Pacific locations.

| CON CLUS ION AND MANAG EMENT IMPLIC ATIONS
This study represents the first genetic investigation of the iconic Galápagos scalloped hammerhead aggregation, and the largest sample size-based investigation of population structure and phylogeography of this species in the ETP to date. Our results, using both matrilineal and nuclear genetic markers, indicate high connectivity between the Galápagos aggregation and all coastal nursery sites, and no convincing evidence of philopatry within the ETP. This extensive connectivity in a region known for high IUU fishing points to the need for coordinated, multinational management cooperation among ETP jurisdictions in order to conserve Sphyrna lewini in both the Galápagos aggregations and ETP as a whole. Additionally, we underscore the need for management strategies for the three evolutionarily unique (phylogeographic) lineages in each ocean basin order to conserve their biodiversity.

CO N FLI C T O F I NTE R E S T
The authors report no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All mitochondrial data underlying our analyses can be accessed